A new method to measure the oxygen affinity of cell-free hemoglobin-based oxygen carriers and cell-free hemoglobin/red blood cell mixtures is under development. This method uses an enzyme to remove oxygen from a hemoglobin solution and calculates saturation via enzyme kinetics. The goal of this Phase I SBIR proposal is to use this method to develop an instrument, which will rapidly provide the oxygen binding curves of concentrated hemoglobin solutions under relevant physiologic conditions. Under specific Aim 1, we will design and instrument a reaction cell to record data from the enzymatic deoxygenation of a hemoglobin solution using the protocatechuic acid (PCA)\protocatechuic acid 3,4-dioxygenase (PCD) system. Enzyme kinetics will be used to determine oxygen content, thus eliminating the need for spectral measurements of hemoglobin saturation. Oxygen equilibrium curve (OEC) measurements will be performed on concentrated hemoglobin solutions or in mixtures with red blood cells. Under specific Aim 2, a Windows-based application will be developed to streamline data acquisition and electrode calibrations. Data analysis will be performed using a stepwise finite-difference scheme, which calculates hemoglobin saturation from the experimental data. This analysis scheme will be implemented in the MATLAB programming environment. Data analysis will be designed in such a way that, in the future, it will be easily incorporated into the Windows application and run on-line. The hypothesis underlying this proposal is that current methods to characterize oxygen affinity are inherently flawed in their inability to analyze physiologically-relevant concentrated hemoglobin solutions, red cell suspensions, or red cell/hemoglobin mixtures, thus creating a need for a new instrument designed specially for the task.